Protein kinases AKT and PKBR1 of Dictyostelium play essential roles for chemotaxis, are phosphorylated and activated by PDK1 and TORC2 kinases, but have different cellular localizing domains19,20. AKT has a PI3K/PIP3-regulated PH domain while PKBR1 is myristoylated and persistently on membranes. We used strains defective for PI3K/PIP3-, PDK1-, and TORC2-signaling or that express phospho-site mutants of AKT and PKBR1 to study their regulations21. Despite certain similarities, AKT and PKBR1 have distinct regulatory paths that impact activation and effector targeting, with PDK1 serving a novel, central role. Activation of AKT/PKBR1 requires phosphorylation by PDK1. Although TORC2 cannot activate AKT/PKBR1, phosphorylation of AKT/PKBR1 by PDK1 requires phosphorylation by TORC2, suggesting regulatory interaction between PDK1/TORC2. We also showed that Dictyostelium PDK1, unlike some other systems, does not require PI3K/PIP3, and, that PKBR1, but not AKT, can be TORC2/PDK1 activated in cells lacking PI3K. Finally, we showed that AKT and PKBR1 exhibit substrate selectivity and identified 2 novel lipid-interacting proteins, preferentially phosphorylated by AKT. These data reveal new regulatory paths upstream and downstream of AKT family kinases. Presenilin (PS) is the catalytic moiety of the g-secretase complex13,14. Inappropriate g-secretase processing of amyloid precursor protein (APP) in humans is associated with familial Alzheimers disease12. Thus, understanding essential elements within each PS/g-secretase component is critical. We identified highly diverged orthologs for each PS/g-secretase component in Dictyostelium, which lacks endogenous APP, Notch, and other characterized PS/&#61543;-secretase substrates22. Nonetheless, WT Dictyostelium accurately processed human APP, while strains deficient in &#61543;-secretase components did not22. We further demonstrated that Dictyostelium require PS/g-secretase components for phagocytosis and cell-fate specification and that regulation of phagocytosis required an active g-secretase, a pathway suggested, but not proven, for mammalian macrophages. Dictyostelium may, therefore, serve to identify novel PS/&#61543;-secretase signaling targets and provide a unique system for high-throughput screening of small molecule libraries for new therapeutics. Stimulation of cAMP receptors in Dictyostelium regulates the activation/de-activation of GSK3, which mediates developmental cell patterning6,7. While Dictyostelium polarize to extracellular cAMP, a potential role for GSK3 in this pathway had not been investigated. We had shown that ZAK1 was an activating tyrosine kinase for GSK3 and have now identified another tyrosine kinase, ZAK2, in the cAMP-activation pathway for GSK323. We found that ZAK2 and ZAK1 separately regulate tyrosine phosphorylation/activation of GSK3 in distinct differentiated cell populations and that ZAK2 acts in both autonomous and non-autonomous pathways to regulate these cell-type differentiations. Finally, we demonstrated that efficient polarization of Dictyostelium toward cAMP depends on ZAK1-mediated tyrosine phosphorylation of GSK3. Combinatorial regulation of GSK3 by ZAK kinases in Dictyostelium guides cell polarity, directional cell migration, and cell differentiation, pathways that extend our understanding of GSK3 signaling throughout the development. We developed miniaturized high-throughput small molecule screens to identify novel regulatory pathways for chemotaxis and development. The assays were validated in 1536-well plate formats by counter screening for toxic compounds and by dose-response analyses using known inhibitors. We screened 4,000 small molecules for inhibitors of chemotaxis and activators and inhibitors of development. Several of the identified compounds are known regulators of Dictyostelium development;other compounds have presumptive targets in mammalian cells, which will assist their biochemical evaluation in Dictyostelium. These large-scale applications will systematically uncover new factors/pathways in Dictyostelium and further explore the potential for shared mechanisms among complex metazoa.